The term “hydrogel” generally connotes a hydrophilic, crosslinked, organic polymeric material (i.e., hydrophilic polymer networks) that swells in and retains water (see, e.g., WO00/66265 and U.S. Pat. No. 6,613,234 (Voute) to Ciphergen Biosystems). Hydrogels have a variety of commercial applications, illustrated by their use in contact lens, sensors, tissue adhesives, drug delivery, dressings, and surface coatings. For example, see U.S. Pat. No. 6,017,577 to Hostettler et al. In particular, hydrogel surface coatings are used in biomedical devices, such as catheters, catheter balloons, and stents, as illustrated by U.S. Pat. No. 5,601,538 to Deem. Hydrogels can be applied as continuous layers or as patterns of discreet regions on a surface (e.g., gel “patches” or “pads”).
The distinctive ability of hydrogels to swell extensively in water, forming a structurally stable but liquid-compatible structure, arises from their lightly crosslinked character, which in turn arises from how they are made. One approach to such manufacture is by photopolymerization and photocrosslinking, respectively, as disclosed in U.S. Pat. No. 5,567,435 and No. 6,156,478. Thus, the '478 patent describes photocrosslinkable and photopatternable hydrogel compositions that are based on an azlactone-functional monomer. These hydrogels can be patterned onto a substrate by means of a photomask or laser-induced thermal imaging, and the azlactone functionality can be used to bind biomolecules to the substrate. According to the '478 patent, the described hydrogel compositions can be used to produce a “microchip,” such as a low- or high-density DNA chip or a microarray of enzyme-containing gel pads.
Another approach to producing hydrogel materials is by deposition of a monomer solution on the substrate surface and in situ polymerization and crosslinking of monomer mixture using a thermal or photoinitiator, as disclosed in PCT application WO 00/66265. Changing the amount of monomer and cross-linker can affect the thickness and pore size of the resulting hydrogel layers.
U.S. Pat. Nos. 5,512,329 and 5,002,582 to Guire et al. discloses polymers which have latent reactive groups for covalent bonding to substrate surfaces. These polymers covalently bond to the substrate surface when the latent reactive groups are stimulated by an external stimulus such as actinic radiation. These polymers, however, are generally designed for repelling protein rather than adsorbing proteins, or selectively interacting with and binding of proteins with tailored control of functional group chemistry. Moreover, these polymers are not prepared by controlled copolymerization methods which allow for suitable hydrogel formation and suitable chemical binding selectivity with proteins and other biomolecules.
Despite their demonstrated versatility and applicability in certain contexts, the potential of hydrogels has not been fully exploited in biochip-based methods of protein detection, in particular, mass-spectral techniques, such as Matrix-Assisted Laser Desorption/Ionization (MALDI) and Surface-Enhanced Laser Desorption/Ionization (SELDI) mass spectroscopy, which are of increasing popularity for protein analysis. Moreover, conventional procedures for producing hydrogels typically do not provide the coating uniformity and homogeneity that would facilitate MALDI or SELDI mass spectroscopy. For example, using in situ polymerization of monomer mixture do not typically provide controlled polymerization processes. The polymerization and surface attachment typically take place simultaneously on an individual spot, and each spot represents a separated reactor. The resulting hydrogel materials can suffer from spot-to-spot and chip-to-chip variations. The conventional procedures also typically do not provide a three-dimensional polymeric structure that has sufficient surface area, controllable porosity and ligand density for capturing proteins and biomolecules in a broad range of molecular weight. The hydrogels having sufficient surface area can provide a probe with high binding capacity and sensitivity, which is attractive when the amount of the sample available for analysis is very small and limited. The hydrogels having controllable pore size and/or ligand density can provide a probe with desirable selectivity and binding capacity that meet the demands of specific biological applications. Also, conventional methods typically do not provide the coating uniformity and homogeneity that would facilitate MALDI or SELDI mass spectroscopy. For instance, uniformity in the hydrogel surface coating may provide a more accurate time-of-flight analysis of samples, as all analytes absorbed on the probe surface are equidistant from an energy source of a gas phase ion spectrometer. Also, conventional formulations for probe materials may not be compatible with desired process methods such as spin coating, dip coating, photopatterning, or useful combinations thereof. The presence of low molecular weight components can cause problems. For instance, see PCT application WO 00/66265 and U.S. patent publication 20020060290 A1. Other literature includes U.S. Pat. No. 6,579,719 (Hutchens), U.S. Pat. No. 6,610,630 (Schwarz), and U.S. Pat. No. 6,675,104 (Paulse).
A need exists to improve biochip methods of detecting biomolecules, such as proteins, including detection by MALDI, SELDI, and other mass-spectrometric analyses through use of probe materials characterized by greater uniformity and structural stability, through better control of coating thickness, hydrogel porosity, and spot variation. Advantages which the present invention provides include maximizing the value of a hydrogel surface for SELDI and MALDI analysis including but not limited to the following factors: (1) complete coverage of the hydrogel, (2) control of hydrogel thickness and swelling degree, (3) uniformity of hydrogel coatings, (4) stability of hydrogel on the surface, (5) controlling the density of the selective binding functionality, (6) ease and consistency of producing hydrogel, and (7) substantially absence of low molecular weight components which can diffuse out and interfere with the analyses by generating signal noise.
Hydrogel blends are known including blends described in U.S. Pat. No. 6,586,493 (Massia), U.S. Pat. No. 6,211,296 (Frate), and U.S. Pat. No. 4,693,887 (Shah). Hydrogel blends, however, are not generally developed for use in mass spectral methods such as, for example, SELDI and MALDI. A need exists to improve the control over the hydrogel system including improved processability, synthetic versatility, economics, and convenience. Challenges exist however because at times the presence of one useful functional group in a hydrogel system can interfere with use of and synthetic strategies for another functional group.